The 43 kd postsynaptic protein (43K) plays a key role in the aggregation of muscle nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane of the neuromuscular junction. By transiently coexpressing 43K and a single AChR subunit (alpha, beta, gamma, or delta) in the quail fibroblast cell line, QT-6, we show that 43K interacts with each subunit to form cell surface clusters in which 43K and receptor subunit are precisely colocalized. Although the level of cell surface expression of single subunits is much lower than that of fully assembled receptor, the clustering of both single subunits and fully assembled AChR occurs efficiently. In addition, 43K-induced clustering is specific for AChR subunits. From these results, we conclude that each pentameric AChR has five potential sites for interacting with 43K during cluster formation.